Investigating Tm Method Specificity Using Oligonucleotide Sequence Variants.

All starting materials and the active pharmaceutical ingredient (API) of a drug product must be subjected to analytical identity (ID) testing as part of the release prior to their introduction into the pharmaceutical manufacturing process. Generally, it is preferable for Quality Control (QC) laboratories to perform ID tests using a simple and fast to perform, yet highly specific, analytical method. This preference also applies to oligonucleotides, an emerging class of APIs, where a combined ID testing strategy should be applied, including intact mass determination and a sequence-specific method. Within this work, we investigated whether ultravioloet (UV)-spectrometric determination of the melting temperature (T_m) of oligonucleotides is a suitable sequence-specific ID test for these substances in the pharmaceutical routine QC. Therefore, this method was evaluated for its specificity toward deviating oligonucleotide sequences. For this, model oligonucleotide sequences and variants thereof were designed, synthesized, and analyzed, resulting in precise and specific data. Even single base exchanges or single nucleotide deletions and insertions in the sequences led to significant changes in the measured T_m of the corresponding oligonucleotide duplexes. These results indicate a generally high specificity of the method toward subtle changes in oligonucleotide sequences and confirm the applicability of the analytical method as part of the ID testing strategy for oligonucleotides in the pharmaceutical QC environment.